PROJECT SUMMARY/ABSTRACT Aggregatibacter actinomycetemcomitans (Aa) is a key pathogen in oral and non-oral infections. Colonization by distinct clonal lineages of Aa may lead to different consequences, from minimal diseases to the development of aggressive periodontitis. The basis for the variable virulence of Aa is not fully understood, and presents a challenge to diagnosis and management of Aa-associated infections. Aa demonstrates significant variation in gene content primarily because of genomic islands, which are associated with bacterial virulence potential. T cell subsets are often targets of microorganisms' virulence mechanisms, whose subversion can affect the course and outcome of infections. The objective of this study is to assess the T cell heterogeneity in the pathogenesis of Aa in biofilm-mediated infection. Our central hypothesis is that the virulence of Aa is modulated by genomic islands and mediated by Th17 cells and counteracted by Treg. To test this hypothesis, we will employ a novel Microfluidic Single-cell Analyzer (MSCA) to characterize the specific T cell subpopulations in rats infected by Aa biofilm in our animal model. Two aims will be pursued: Aim 1. Optimize single-cell molecular profiling of Th17 and Treg subsets in uninfected Rats. We will optimize the device for processing flow cytometrically-isolated Th17 and Treg subsets of normal lymph node cells. Single-cell transcriptomes of Th17 and Treg will be obtained with MSCA and RNA-seq and analyzed to determine the background heterogeneity of Th17 and Treg subsets in uninfected Rats. Aim 2. Assess cellular heterogeneity of Th17 and Treg in Aa biofilm-infected Rats. Single-cell transcriptomes of Th17 and Treg from rats infected with Aa wildtype strains and island-deletion mutants will be obtained with MSCA. These transcriptomes of the cells will be clustered by similarity to reveal heterogeneity of Th17 and Treg. Sub-populations (clusters of similar single cell transcriptomes) of Th17 and Treg will be isolated with their unique profile of mediators from rats with Aa pathogenesis. The outlined studies will evaluate and identify virulence-associated genomic islands of Aa and the corresponding protective or destructive immune responses mediated by Th17 and Treg. The results are expected to have an important positive impact, because the information will expand the knowledge to the pathogenesis of Aa leading to improved diagnostics and treatment of oral biofilm infections.